The immune response to microbial pathogens is initiated by recognition of specific pathogen components by host cells both at the cell surface and in the cytosol. While the response triggered by pathogen products at the surface of immune cells is well characterized, that initiated in the cytosol is poorly understood. Nod1 is a member of a growing family of proteins with structural homology to apoptosis regulators Apaf-1/Ced-4 and plant disease resistant R gene products. Nod1 promotes apoptosis when overexpressed in cells, but unlike Apaf-1, it induces NF-kappaB activation. NF-KappaB activation induced by Nod1 is mediated by the association of the CARD of Nod1 with the corresponding CARD of RICK, a protein kinase that activates NF-kappaB. Analyses with wild-type (wt) and mutant forms of both Nod1 and RICK have suggested that Nod1 and RICK act in the same pathway of NF-kappaB activation, where RICK functions as a downstream mediator of Nod1 signaling. Nod1 self-associates through its nucleotide-binding domain (NBD) and Nod1 oligomerization promotes proximity of RICK molecules and NF-kappaB activation. Like Nod1, intracellular plant R proteins contain N-terminal effector domains linked to a NBD and multiple leucine-rich repeats (LRRs) located C-terminally of the NBD. The LRRs of R proteins are highly diverse and appear to be involved in the recognition of a wide array of pathogen components. Remarkably, we find that bacterial lipopolysacharides (LPS), but not other pathogen components, induced TLR4- and MyD88- independent NF-kappaB activation in human embryonic kidney 293T cells expressing trace amounts of Nod1. Like plant disease resistant R proteins, the LRRs of Nod1 were required for LPS-induced NF-kappaB activation. Furthermore, a LPS binding activity could be co-immunopurified with Nod1 from cytosolic extracts. Our hypothesis is that Nod1 is a mammalian counterparts of plant R gene products that may function as a cytosolic receptor for pathogen components derived from invading bacteria. We propose three Specific Aims to understand the biochemical mechanism by which bacterial LPS and Nod1 interact and to determine the role of Nod1 in the response to bacterial LPS in vivo.